This invention relates to methods of screening molecules capable of inhibiting the survival of Helicobacter, particularly Helicobacter pylori, in vivo by specifically, inhibiting the activity of UreI, to the molecules identified by these methods, and to the use of these molecules to treat or prevent Helicobacter infection.
Helicobacter priori is a microaerophilic Gram-negative bacterium, which colonizes the gastric mucosa of humans (10). H. pylori is associated with gastritis and peptic ulcer disease and has been shown to increase the risk of gastric cancers. Urease is a major virulence factor of H. pylori. It is involved in neutralizing the acidic microenvironment of the bacterium and also plays a role in H. pylori metabolism (11, 26).
The urease region of the H. pylori genome is composed of two gene clusters common to all strains (9 and FIG. 1), one comprising the ureAB genes encoding the structural urease subunits and the other containing the ureEFGH genes encoding the accessory proteins required for nickel incorporation into the urease active site. The UreI gene lies immediately upstream from this latter gene cluster and is transcribed in the same direction (FIG. 1). The ureA, ureB, ureE, ureF, ureG, ureH, and ureI genes and gene products have been described and claimed in U.S. Pat. No. 5.695,931 and allowed patent application Ser. No. 08/472,285. both of which are specifically incorporated herein by reference.
The distances separating UreI from ureE (one base pair, bp) and ureE from ureF (11 bp) suggest that ureI-ureE-ureF constitute an operon. Cotranscription of ureI and ureE has been demonstrated by northern blot analysis (1). An H. pylori N6 mutant with a ureI gene disrupted by a MiniTn3-Km transposon was previously described by Ferrero et al. (1994) (13). This strain (N6-ureI::TnKm-8) presented a urease negative phenotype, so it was concluded that ureI was an accessory gene required for full urease activity.
The sequences of UreI from H. pylori and the AmiS proteins, encoded by the aliphatic amidase operons of Pseudomonas aeruginosa and Rhodococcus sp. R312, are similar (5, 27). Aliphatic amidases catalyze the intracellular hydrolysis of short-chain aliphatic amides to produce the corresponding organic acid and ammonia. It has been shown that H. pylori also has such an aliphatic amidase, which hydrolyzes acetamide and propionamide in vitro (23).
In view of the sequence similarity between UreI and AmiS together with the very similar structures of the urease and amidase substrates (urea: NH2xe2x80x94COxe2x80x94NH2 and acetamide: CH3xe2x80x94COxe2x80x94NH2) and the production of ammonia by both enzymes, a better understanding of the function of the H. pylori UreI protein is required. This understanding will open new opportunities for the prevention and treatment of H. pylori infections.
This invention provides methods for identifying molecules capable of inhibiting the growth and/or survival of Helicobacter species, particularly, H. pylori, in vivo. In particular, the methods of this invention involve screening molecules that specifically inhibit UreI protein function.
The invention encompasses the molecules identified by the methods of this invention and the use of the molecules by the methods of this invention to treat or prevent Helicobacter, and particularly H. pylori, infection in humans and animals.
Another aspect of this invention is a method of preventing or treating Helicobacter species infection by administration to a human or animal in need of such treatment a molecule capable of inhibiting the growth and/or survival of Helicobacter species in vivo. One such molecule according to the invention is characterized by a high affinity for UreI, which allows it (i) to be transported inside the Helicobacter cell, or (ii) to inhibit transport properties of UreI, or (iii) to inhibit UreI function by inhibiting UreI interaction with urease or other Helicobacter proteins. By inhibiting UreI, such molecule renders the bacteria more sensitive to acidity.
Yet another aspect of this invention is the production of immunogenic UreI antigens and their use as vaccines to prevent Helicobacter species infection and/or colonization of the stomach or the gut. Antibodies to these UreI antigens are also encompassed within the scope of this invention.
This invention further relates to recombinant strains of H. pylori comprising a modified ureI gene, such that the products of the modified gene contribute to the attenuation of the bacteria""s ability to survive in vivo and thus, its pathogenic effects.